PROJECT SUMMARY The proposed work seeks to uncover novel mechanisms governing the assembly of the extracellular matrix (ECM) protein fibronectin (FN) and its role in directing assembly of other ECM proteins to form a definitive matrix. Because the definitive matrix is integral to key physical, chemical, and mechanical properties that regulate tissue structures and functions, understanding mechanisms that coordinate the assembly of FN with type I collagen and other ECM proteins is crucial. Molecular defects in matrix assembly are implicated in fibrosis in which disordered ECM fibers accumulate due to abnormal production of FN, collagen I, and other matrix proteins. Along with fibrosis, skeletal abnormalities, tumorigenesis, and other ECM-related diseases affect millions of people around the globe yet in most cases the ECM defects are poorly understood and, in many ways, still largely untreatable. The proposed work will test the hypothesis that the organization and insolubility of the pericellular FN matrix control and direct the assembly of a tissue-appropriate definitive matrix, and that perturbation of the FN matrix disrupts tissue and cell functions leading to disease. We have a general understanding of the main steps of FN matrix assembly, but specific mechanisms governing FN fibril organization, fibril stability, or how FN guides assembly of collagens have yet to be elucidated. The proposed aims will address these mechanisms, building on the foundation that we have established with our previous work. We will use our proven matrix assembly systems to analyze the formation of FN fibrils and their contributions to type I collagen assembly to determine the protein interactions that are critical for directing definitive matrix assembly. The goal of Aim 1 is to determine the mechanism that converts reversible FN-FN interactions into stable insoluble fibrils. We will test a new hypothesis that heparin/heparan sulfate acts as a molecular switch by binding to FN and inducing conformational changes that promote strong protein-protein interactions. Aim 2 will address the hypothesis that FN matrix acts as a template for collagen fibrillogenesis by providing a platform for the localization and activation of collagen processing enzymes. Aim 3 will link FN assembly with cell differentiation. Using a newly discovered human FN mutation potentially linked to a skeletal dysplasia, we will determine the matrix assembly defect caused by this mutation and will apply the micromass chondrogenesis model system to understand the effects of mutant FN matrix on cell differentiation. Our work will fill critical knowledge gaps in our understanding of the mechanisms governing key steps in the assembly of a definitive matrix, and will suggest routes by which pathological processes or mutations can cause abnormal matrix organization or accumulation. This work will generate new ideas for strategies to manipulate ECM assembly in order to treat or control fibrosis and other ECM-related diseases and may lead to ECM-based treatments to improve disease outcomes.